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IntroductionBacteria inhabit the in- and outside of the human body, such as skin, gut or the oral cavity where they play an innoxious, beneficial or even pathogenic role. It is well known that bacteria can secrete membrane vesicles (MVs) like eukaryotic cells with extracellular vesicles (EVs). Several studies indicate that bacterial membrane vesicles (bMVs) play a crucial role in microbiome-host interactions. However, the composition of such bMVs and their functionality under different culture conditions are still largely unknown.MethodsTo gain a better insight into bMVs, we investigated the composition and functionality of E. coli (DSM 105380) bMVs from the culture media Lysogeny broth (LB) and RPMI 1640 throughout the different phases of growth (lag-, log- and stationary-phase). bMVs from three time points (8 h, 54 h, and 168 h) and two media (LB and RPMI 1640) were isolated by ultracentrifugation and analyzed using nanoparticle tracking analysis (NTA), cryogenic electron microscopy (Cryo-EM), conventional transmission electron microscopy (TEM) and mass spectrometry-based proteomics (LC–MS/MS). Furthermore, we examined pro-inflammatory cytokines IL-1β and IL-8 in the human monocyte cell line THP-1 upon bMV treatment.ResultsParticle numbers increased with inoculation periods. The bMV morphologies in Cryo-EM/TEM were similar at each time point and condition. Using proteomics, we identified 140 proteins, such as the common bMV markers OmpA and GroEL, present in bMVs isolated from both media and at all time points. Additionally, we were able to detect growth-condition-specific proteins. Treatment of THP-1 cells with bMVs of all six groups lead to significantly high IL-1β and IL-8 expressions.ConclusionOur study showed that the choice of medium and the duration of culturing significantly influence both E. coli bMV numbers and protein composition. Our TEM/Cryo-EM results demonstrated the presence of intact E. coli bMVs. Common E. coli proteins, including OmpA, GroEL, and ribosome proteins, can consistently be identified across all six tested growth conditions. Furthermore, our functional assays imply that bMVs isolated from the six groups retain their function and result in comparable cytokine induction.

ABSTRACT Vesiculation is a process employed by Gram-negative bacteria to release extracellular vesicles (EVs) into the environment. EVs from pathogenic bacteria play functions in host immune modulation, elimination of host defenses, and acquisition of nutrients from the host. Here, we observed EV production of the bacterial speck disease causal agent, Pseudomonas syringae pv. tomato (Pto) DC3000, as outer membrane vesicle release. Mass spectrometry identified 369 proteins enriched in Pto DC3000 EVs. The EV samples contained known immunomodulatory proteins and could induce plant immune responses mediated by bacterial flagellin. Having identified two biomarkers for EV detection, we provide evidence for Pto DC3000 releasing EVs during plant infection. Bioinformatic analysis of the EV-enriched proteins suggests a role for EVs in antibiotic defense and iron acquisition. Thus, our data provide insights into the strategies this pathogen may use to develop in a plant environment. IMPORTANCE The release of extracellular vesicles (EVs) into the environment is ubiquitous among bacteria. Vesiculation has been recognized as an important mechanism of bacterial pathogenesis and human disease but is poorly understood in phytopathogenic bacteria. Our research addresses the role of bacterial EVs in plant infection. In this work, we show that the causal agent of bacterial speck disease, Pseudomonas syringae pv. tomato, produces EVs during plant infection. Our data suggest that EVs may help the bacteria to adapt to environments, e.g., when iron could be limiting such as the plant apoplast, laying the foundation for studying the factors that phytopathogenic bacteria use to thrive in the plant environment.

This study aimed to determine the metabolic response of growing German Simmental bulls fed rations low in crude protein (CP) supplemented with rumen-protected methionine (RPMET). In total, 69 bulls (on average 238 ± 11 days of age at start and 367 ± 25 kg of bodyweight) were assigned to three dietary treatments (n = 23/group): Positive control (CON; 13.7% CP; 2.11 g methionine/kg DM), negative control deficient in CP (RED; 9.04% CP; 1.56 g methionine/kg DM) and crude protein-deficient ration supplemented with RPMET (RED+RPMET; 9.04% CP; 2.54 g methionine/kg DM). At slaughter, samples of liver, muscle and blood serum were taken and underwent subsequent metabolomics profiling using a UHPLC-QTOF-MS system. A total of 6540 features could be detected. Twenty metabolites in the liver, five metabolites in muscle and thirty metabolites in blood serum were affected (p < 0.05) due to dietary treatments. In total, six metabolites could be reliably annotated and were thus subjected to subsequent univariate analysis. Reduction in dietary CP had minimal effect on metabolite abundance in target tissues of both RED and RED+RPMET bulls as compared to CON bulls. The addition of RPMET altered the hepatic anti-oxidant status in RED+RPMET bulls compared to both RED and CON bulls. Results exemplify nutrient partitioning in growing German Simmental bulls: bulls set maintenance as the prevailing metabolic priority (homeostasis) and nutrient trafficking as the second priority, which was directed toward special metabolic functions, such as anti-oxidant pathways.

Animal farms have become one of the most important reservoirs of carbapenem-resistant Klebsiella spp. (CRK) owing to the wide usage of veterinary antibiotics. “One Health”-studies observing animals, the environment, and humans are necessary to understand the dissemination of CRK in animal breeding areas. Based on the concept of “One-Health,” 263 samples of animal feces, wastewater, well water, and human feces from 60 livestock and poultry farms in Shandong province, China were screened for CRK. Five carbapenem-resistant Klebsiella pneumoniae (CRKP) and three carbapenem-resistant Klebsiella quasipneumoniae (CRKQ) strains were isolated from animal feces, human feces, and well water. The eight strains were characterized by antimicrobial susceptibility testing, plasmid conjugation assays, whole-genome sequencing, and bioinformatics analysis. All strains carried the carbapenemase-encoding gene blaNDM-5, which was flanked by the same core genetic structure (IS5-blaNDM-5-bleMBL-trpF-dsbD-IS26-ISKox3) and was located on highly related conjugative IncX3 plasmids. The colistin resistance gene mcr-8.1 was carried by three CRKP and located on self-transmissible IncFII(K)/IncFIA(HI1) and IncFII(pKP91)/IncFIA(HI1) plasmids. The genetic context of mcr-8.1 consisted of IS903-orf-mcr-8.1-copR-baeS-dgkA-orf-IS903 in three strains. Single nucleotide polymorphism (SNP) analysis confirmed the clonal spread of CRKP carrying-blaNDM-5 and mcr-8.1 between two human workers in the same chicken farm. Additionally, the SNP analysis showed clonal expansion of CRKP and CRKQ strains from well water in different farms, and the clonal CRKP was clonally related to isolates from animal farms and a wastewater treatment plant collected in other studies in the same province. These findings suggest that CRKP and CRKQ are capable of disseminating via horizontal gene transfer and clonal expansion and may pose a significant threat to public health unless preventative measures are taken.

Tobacco bacterial wilt caused by Ralstonia solanacearum is one of the most devastating diseases. Microbial keystone taxa were proposed as promising targets in plant disease control. In this study, we obtained an antagonistic Bacillus isolate EM-1 from bacterial wilt-suppressive soil, and it was considered rhizosphere-resident bacteria based on high (100%) 16S rRNA gene similarity to sequences derived from high-throughput amplicon sequencing. According to 16S rRNA gene sequencing and MLSA, strain EM-1 was identified as Bacillus velezensis. This strain could inhibit the growth of R. solanacearum, reduce the colonization of R. solanacearum in tobacco roots, and decrease the incidence of bacterial wilt disease. In addition, strain EM-1 also showed a strong inhibitory effect on other phytopathogens, such as Alternaria alternata and Phytophthora nicotianae, indicating a wide antagonistic spectrum. The antimicrobial ability of EM-1 can be attributed to its volatile, lipopeptide and polyketide metabolites. Iturin A (C14, C15, and C16) was the main lipopeptide, and macrolactin A and macrolactin W were the main polyketides in the fermentation broth of EM-1, while heptanone and its derivatives were dominant among the volatile organic compounds. Among them, heptanones and macrolactins, but not iturins, might be the main potential antibacterial substances. Complete genome sequencing was performed, and the biosynthetic gene clusters responsible for iturin A and macrolactin were identified. Moreover, strain EM-1 can also induce plant resistance by increasing the activity of CAT and PPO in tobacco. These results indicated that EM-1 can serve as a biocontrol Bacillus strain for tobacco bacterial wilt control. This study provides a better insight into the strategy of exploring biocontrol agent based on rhizosphere microbiome.

ABSTRACT The long-term exposure to enclosed environments may lead to chronic stress in crewmembers and affect their physical and mental state. Salivary microbiome and biomarkers of immune function are increasingly used in human health research. The “Lunar Palace 365” project, which was a 370-day, multicrew, enclosed experiment carried out in a ground-based bioregenerative life support system platform named Lunar Palace 1 (LP1). We investigated the temporal dynamics of the salivary microbiota and cytokines in the third phase of the “Lunar Palace 365” experiment, including 1 month before entering LP1 and 1 month after leaving Lp1. Results reveal no regular temporal change pattern in these parameters (highly abundant phyla and genera) during the experiment. Although the crewmembers’ oral microbiota temporally changed, it recovered quickly after the study subjects left the enclosed environment. The levels of IL-6, IL-10, and TNF-α in crewmembers’ saliva decreased after leaving the normal environment for the enclosed environment, indicating that their oral inflammatory response level was reduced. There were significant individual differences in crewmembers’ salivary microbiota, however, the shared living space reduced these differences. Moreover, air microbiota might have also played a significant role in reducing the individual differences. In summary, the enclosed environment did not result in persistent changes in human salivary microbiota and oral immunity. This study provides some insights for studying the effect of enclosed controlled environments on human immunity and microbiome. IMPORTANCE Long-term exposure to space environments may influence the human microbiome, the human immune system, and the intricate balance between the two, causing impaired immunity and increased disease susceptibility. It was previously believed that the main potential factors of long-term spaceflight on human health were microgravity and radiation. However, the effects of long-term enclosed environments on human health were unclear. Bioregenerative life support systems (BLSS) is a good experimental model for studying the effects of enclosed environments on human systemic microbiota and immune disorders. We monitored the microbiota and cytokines in the saliva of crewmembers before they entered BLSS, during their stay in BLSS, and after leaving BLSS. The results indicated long-term closed environment will not cause persistent changes in human salivary microbiota and immunity.

ABSTRACT Salinity is an important abiotic stress affecting plant growth. We have known that plants can recruit beneficial microbes from the surrounding soil. However, the ecological functions of the core microbiome in salt-tolerant plants, together with their driving factors, remain largely unexplored. Here, we employed both amplicon and shotgun metagenomic sequencing to investigate the microbiome and function signatures of bulk soil and rhizocompartment samples from three salt-tolerant plants (legumes Glycine soja and Sesbania cannabina and nonlegume Sorghum bicolor). Strong filtration effects for microbes and functional genes were found in the rhizocompartments following a spatial gradient. The dominant bacteria belonged to Ensifer for legumes and Bacillus for S. bicolor. Although different salt-tolerant plants harbored distinct bacterial communities, they all enriched genes involved in cell motility, Na+ transport, and plant growth-promoting function (e.g., nitrogen fixation and phosphate solubilization) in rhizoplane soils, implying that the microbiome assembly of salt-tolerant plants might depend on the ecological functions of microbes rather than microbial taxa. Moreover, three metagenome-assembled genomes affiliated to Ensifer were obtained, and their genetic basis for salt stress alleviation were predicted. Soil pH, electrical conductivity, and total nitrogen were the most important driving factors for explaining the above microbial and functional gene selection. Correspondingly, the growth of an endophyte, Ensifer meliloti CL09, was enhanced by providing root exudates, suggesting that root exudates might be one of factors in the selection of rhizosphere and endosphere microbiota. Overall, this study reveals the ecological functions of the populations inhabiting the root of salt-tolerant plants. IMPORTANCE Salinity is an important but little-studied abiotic stressor affecting plant growth. Although several previous reports have examined salt-tolerant plant microbial communities, we still lack a comprehensive understanding about the functional characteristics and genomic information of this population. The results of this study revealed the root-enriched and -depleted bacterial groups, and found three salt-tolerant plants harbored different bacterial populations. The prediction of three metagenome-assembled genomes confirmed the critical role of root dominant species in helping plants tolerate salt stress. Further analysis indicated that plants enriched microbiome from soil according to their ecological functions but not microbial taxa. This highlights the importance of microbial function in enhancing plant adaptability to saline soil and implies that we should pay more attention to microbial function and not only to taxonomic information. Ultimately, these results provide insight for future agriculture using the various functions of microorganisms on the saline soil.

Soil salinization limits crop growth and yield in agro-ecosystems worldwide by reducing soil health and altering the structure of microbial communities. Salt-tolerant plant growth-promoting rhizobacteria (PGPR) alleviate plant salinity stress. Wild soybean (Glycine soja Sieb. and Zucc.) is unique in agricultural ecosystems owing to its ability to grow in saline-alkali soils and fix atmospheric nitrogen via symbiotic interactions with diverse soil microbes. However, this rhizosphere microbiome and the nodule endosymbionts have not been investigated to identify PGPR. In this study, we investigated the structural and functional rhizosphere microbial communities in saline-alkali soil from the Yellow River Delta and coastal soil in China, as well as wild soybean root nodule endosymbionts. To reveal the composition of the microbial ecosystem, we performed 16S rRNA and nifH gene amplicon sequencing on root nodules and root zones under different environmental conditions. In addition, we used culture-independent methods to examine the root bacterial microbiome of wild soybean. For functional characterization of individual members of the microbiome and their impact on plant growth, we inoculated isolates from the root microbiome with wild soybean and observed nodulation. Sinorhizobium/Ensifer accounted for 97% of the root nodule microbiome, with other enriched members belonging to the phyla Actinobacteria, Bacteroidetes, Chloroflexi, Acidobacteria, and Gemmatimonadetes; the genera Sphingomonas, Microbacterium, Arthrobacter, Nocardioides, Streptomyces, Flavobacterium, Flavisolibacter, and Pseudomonas; and the family Enterobacteriaceae. Compared to saline-alkali soil from the Yellow River Delta, coastal soil was highly enriched for soybean nodules and displayed significant differences in the abundance and diversity of β-proteobacteria, δ-proteobacteria, Actinobacteria, and Bacteroidetes. Overall, the wild soybean root nodule microbiome was dominated by nutrient-providing Sinorhizobium/Ensifer and was enriched for bacterial genera that may provide salt resistance. Thus, this reductionist experimental approach provides an avenue for future systematic and functional studies of the plant root microbiome.

Besides intrinsic and extrinsic factors such as antagonism for organic substrates or temperature, the storage atmosphere of meat has a high influence on the development of its initial microbiota. Specific modified atmospheres (MAs) selectively suppress growth of aerobic and anaerobic bacteria, thus reshaping the initial microbiota. As some microorganisms are more tolerant to MA, they overgrow competitors and produce metabolites that cause rejection of the product. In order to elucidate responses to different MA by means of metabolic adaptation and competition for organic substrates on meat, the typical representative meat spoilage bacteria Brochothrix (B.) thermosphacta TMW2.2101 and four lactic acid bacteria Carnobacterium (C.) divergens TMW2.1577, C. maltaromaticum TMW2.1581, Leuconostoc (L.) gelidum subsp. gelidum TMW2.1618 and L. gelidum subsp. gasicomitatum TMW2.1619 were chosen. Bacteria were grown in sterile glass bottles filled with a meat simulation medium, which was aerated constantly with either air, 100%_N2, 30%_CO2/70%_O2 or 30%_CO2/70%_N2. Growth of bacteria during incubation at 25°C and stirring at 120 rpm was monitored over 48 h and a label-free quantitative mass spectrometric approach was employed to determine changes within the bacterial proteomes in response to oxygen and carbon dioxide. Both Leuconostoc subsp. were intrinsically tolerant to MA, exhibiting no proteomic regulation of enzymes, whereas the other species provide a set of metabolic adaptation mechanism, enabling higher resistance to the detrimental effects of MA. Those mechanisms comprise: enhanced oxidative stress reduction, adjustment of the pyruvate metabolism and catabolic oxygen consumption in response to oxygen and intracellular pH homeostasis, maintenance of osmotic balance and alteration of the fatty acid composition in response to carbon dioxide. We further evaluated the potential of industrial used MA to inhibit specific bacterial spoilage. No bacterial inhibition is predicted for 30%_CO2/70%_O2 for the analyzed species, whereas 30%_CO2/70%_N2 predictively inhibits C. divergens TMW21577 and B. thermosphacta TMW2.2101. Furthermore, species-specific metabolic pathways enabling different and preferential carbon source utilization were identified, which enable non-competitive coexistence of respective bacteria on meat, resulting in synergistic spoilage. In conclusion, this study gives mechanistically explanations of their acknowledged status as typical spoilage organisms on MAP meats.

In this paper, we address the lack of specific targeting of β-amyloid β-protein (Aβ) in the current Alzheimer's disease (AD) diagnosis and treatment, and build a novel AIE nanoprobe molecule based on molecular design and nano-self-assembly technology. Based on the molecular design and nano-self-assembly technology, a new two-component AIE nanoprobe molecule with integrated AD diagnosis and treatment was constructed by co-assembling two-component AIE components. The NIR AIE nanomolecule can effectively penetrate the blood brain barrier (BBB) and depolymerize Aβ fibers, alleviate reactive oxygen species (ROS) in the focal area, and achieve highly sensitive imaging and specific depolymerization.

Death Associated Protein Kinase 1 (DAPK1) is an important signaling kinase mediating the biological effect of multiple natural biomolecules such as IFN-γ, TNF-α, curcumin, etc. DAPK1 is degraded through both ubiquitin-proteasomal and lysosomal degradation pathways. To investigate the crosstalk between these two DAPK1 degradation pathways, we carried out a screen using a set of ubiquitin E2 siRNAs at the presence of Tuberous Sclerous 2 (TSC2) and identified that the small ubiquitin-like molecule (SUMO) pathway is able to regulate the protein levels of DAPK1. Inhibition of the SUMO pathway enhanced DAPK1 protein levels and the minimum domain of DAPK1 protein required for this regulation is the kinase domain, suggesting that the SUMO pathway regulates DAPK1 protein levels independent of TSC2. Suppression of the SUMO pathway did not enhance DAPK1 protein stability. In addition, mutation of the potential SUMO conjugation sites on DAPK1 kinase domain did not alter its protein stability or response to SUMO pathway inhibition. These data suggested that the SUMO pathway does not regulate DAPK1 protein degradation. The exact molecular mechanism underlying this regulation is yet to be discovered.